Hepatitis C virus (HCV) infects approximately 130 million people worldwide and poses a major threat to human health. HCV is a hepatotropic RNA virus that causes chronic hepatic inflammation in ~70% of infected individuals, putting patients at risk of developing cirrhosis and hepatocellular carcinoma. Clearance of HCV infection requires the development of vigorous and broad CD4 and CD8 T cell responses. Unfortunately, these responses often fail and are substituted with an intermediate cytotoxic T cell response unable to eliminate the infection but strong enough to cause hepatocyte destruction. The innate immune responses to HCV provide the first line of defense against the virus and are pivotal for orchestrating subsequent T cell responses, but how they operate in hepatocytes remains poorly understood. Our recent data suggest that human hepatocytes contain a functional Toll-like receptor-3 (TLR3) signaling pathway that senses HCV infection and leads to production of proinflammatory cytokines and chemokines which are central to recruiting T cells to the liver. However, the mechanisms by which TLR3 senses HCV double-stranded (ds) RNAs generated during viral replication and results in NF-kB activation and subsequent induction of proinflammatory mediators are unclear. We hypothesize that these molecular events in HCV-hepatocyte interactions are crucial in host immune responses to HCV. Therefore a better understanding of these molecular events is crucial for advancing our knowledge on host immune responses to HCV and the mechanisms of TLR3 signaling in hepatocytes, and lays the foundation for developing novel immunotherapies for hepatitis C. The work proposed involves two specific aims. Aim 1 will determine the mechanism by which HCV dsRNAs generated during viral replication are sensed by TLR3 in endosome/lysosome compartments. We will delineate the role of autophagy in TLR3- dependent host response to HCV infection, and identify and characterize cellular proteins that are recruited to endolysosomes to facilitate TLR3 signaling during HCV infection. Aim 2 will characterize the signaling mechanisms downstream of the TLR3 pathway by which HCV activates NF-kB and subsequent induction of proinflammatory cytokines and chemokines. We will also determine the expression of selected innate immune signaling molecules and proinflammatory mediators in diagnostic liver biopsy specimens from a specific cohort of hepatitis C patients and define their possible relationship to hepatic inflammation.